1. Field of the Invention
The invention provides Mycobacterium strains that have an enhanced ability to elicit an immune response. In vivo experiments have demonstrated, for example, a Major Histocompatibility Complex Class I-restricted CD8+ T cell immune response. In particular, the invention provides Mycobacterium strains that express a Perfringolysin O (PfoA) protein that permits escape of the Mycobacterium from endosomes, and vaccine preparations containing the Mycobacterium strains.
2. Background
Mycobacterium tuberculosis (M. tb) has infected one-third of the world's population, causing active disease in 8 million and killing 1.6-2.2 million individuals every year, most of whom live in the developing world. Tuberculosis (TB) is an epidemic of global proportions that is growing and becoming even more deadly as it intersects with the spread of HIV. TB is the number one killer of people with AIDS.
Bacille Calmette Guerin (BCG), an attenuated strain of Mycobacterium bovis and the current widely used TB vaccine, was developed over 80 years ago and when tested has had widely variable rates of efficacy against pulmonary tuberculosis, including no efficacy in the last large field trial conducted in India (Fine et al., Vaccine, 16(20):1923-1928; 1998; Anonymous, Indian J Med. Res., August; 110:56-69; 1999. Nonetheless, The World Health Organization currently recommends BCG at birth or first contact with health services for all children (except those with symptoms of HIV disease/AIDS) in high TB prevalent countries. This policy is based on evidence that BCG protects against serious childhood forms of TB (Lanckriet et al., Int J Epidemiol, 24(5):1042-1049; 1995; Rodrigues et al., J. Epidemiol Community Health 45(1): 78-80; 1991. Protection by BCG against TB beyond early childhood is a controversial subject with limited data giving mixed results. The high incidence of pediatric and adult TB in developing countries where infant BCG immunization is widely practiced, however, indicates that BCG as currently administered is not highly efficacious over the many years when people are at risk of TB disease. Thus, BCG is considered to be an inadequate public health tool for the intervention and control of TB.
Approximately 70 percent of humans exposed to TB organisms, and who have normal immune systems, do not become infected, and of those that do become infected only about 5 percent develop disease within the first two years. The majority of infected individuals suppress the infection, which is associated with the development of robust cellular immune responses to M. tb antigens. An additional 5 percent later reactivate when immunity declines. Both primary and reactivation disease are much more common in people with HIV/AIDS, again emphasizing the role of immunity in preventing and controlling infection.
Because most humans are able to control TB, there is good reason to hope that by inducing long lasting immunity of the appropriate kind, it should be possible to develop effective vaccines that prevent initial infection after exposure, prevent early progression to disease, prevent reactivation from the latent state, and prevent relapse after treatment. Ultimately, it is the combination of systematic vaccine use plus chemotherapeutic intervention that will eventually eliminate M. tb as a human pathogen.
In light of the critical role childhood BCG vaccination is thought to play in preventing acute TB, it is difficult to replace BCG in trials to evaluate candidate TB vaccines without overwhelming evidence that the new TB vaccine is a superior product. The problem is that M. tb is a human-specific pathogen and animal models only mimic parts of the host-pathogen interaction. Thus, definitive evidence that a new TB vaccine possesses improved potency can only be obtained from controlled field trials in humans. This reality has led many investigators to conclude that a key step toward an improved TB vaccine will be to enhance the immunogenicity of BCG.
One example of such a strategy is to improve the capacity of BCG to induce or activate T cells for enhancing the immune response. The pivotal role of major histocompatibility complex class I-restricted CD8+ T cells in immunity to M tb is demonstrated by the failure of β2-microglobulin (β2m)-deficient mice to control experimental M. tb infection (Flynn et al., PNAS USA, 89(24): 12013-12017; 1992). The pivotal role of Major Histocompatibility Complex class I-restricted CD8+ T cells was convincingly demonstrated by the failure of β2-microglobulin (β2m) deficient mice to control experimental M. tuberculosis infection (Flynn et al., supra, 1992). Because these mutant mice lack Major Histocompatibility Complex class I, functional CD8+ T cells cannot develop. In contrast to M. tuberculosis infection, β2m-deficient mice are capable of controlling certain infectious doses of the BCG vaccine strain (Flynn et al., supra, 1992; Ladel C. H., et al., Eur J Immunol, 25:377-384; 1995). Furthermore, BCG vaccination of β2m-deficient mice only prolonged survival after M. tuberculosis infection, whereas BCG-immunized C57BL/6 resisted M. tuberculosis (Flynn et al., supra, 1992).
This differential CD8+ T cell dependency between M. tuberculosis and BCG may be explained as follows: M. tuberculosis antigens gain better access to the cytoplasm than antigens from BCG leading to more pronounced Major Histocompatibility Complex class I presentation (Hess and Kaufmann, FEMS Microbiol. Immunol 7:95-103; 1993). Consequently, a more effective CD8+ T cell response is generated by M. tuberculosis. This notion was recently supported by increased Major Histocompatibility Complex class I presentation of an irrelevant antigen, ovalbumin, by simultaneous M. tuberculosis, rather than BCG, infection of antigen presenting cells (APC) (Mazzaccaro et al., Proc Natl Acad Sci USA, October 15:93(21):11786-91; 1996).
Thus, M. tb antigens access the host cell cytoplasm better than antigens from BCG, leading to increased Major Histocompatibility Complex (herein referred to as MHC) class I presentation (Hess et al., supra, 1993) and an elevated CD8+ T cell response to M. tb. Further, M. tb stimulates antigen-specific MHC class II-restricted CD4+ T helper cells as well as Major Histocompatibility Complex class I-restricted CD8+ cytotoxic T cells in mice and humans (Kaufmann, Annu Rev Immunol 11:129-163; 1993). By extension, this fact indicates that M tb infected cells are susceptible to recognition by MHC class I-restricted CD8+ cytotoxic T cells. Given that 70 percent of immunocompetent humans exposed to TB organisms do not become infected, immunity induced by M. tb infection is highly effective in controlling this organism in the vast majority of instances. It is believed, therefore, that the efficacy of existing TB vaccine strain BCG will be improved by increasing the capacity of BCG to induce MHC class I-restricted CD8+ cytotoxic T cell responses (Kaufmann, Fundamental Immunology, 1997).
As a rule, antigens expressed by pathogens that remain phagosome-bound are primarily presented by MHC class II molecules to CD4+ T cells but are poorly recognized by CD8+ T cells, which normally recognize antigens presented in the context of MHC class I molecules (Kaufmann, supra, 1997). In contrast, intracellular bacteria, such as Listeria monocytogenes (e.g. ATCC #13932), that escape the phagosome and replicate in the cytoplasm of host cells are effective at accessing the MHC class I antigen presentation pathway and at eliciting CD8+ T cell responses (Berche et al., J Immunol, 138:2266-2276; 1987). This endosome escape function of Listeria monocytogenes was recently transferred into attenuated Salmonella, which normally resides in the phagosome, by introducing the sequences encoding listeriolysin (Llo); the resultant strains were shown to escape the endosome and were more effective at inducing CD8+ T cell responses (Bielecki et al., Nature (London), 354:175-176; 1990; Gentschev et al., Infect Immun 63(10):4202-4205; 1995; Hess et al., Host Response to Intracellular Pathogens, 75-90; 1997). More recently this approach was applied to BCG; thus rBCG strains secreting Llo were constructed to improve the capacity of BCG to induce MHC class I-restricted immune responses (Hess et al., PNAS USA, 95(9):5299-5304; 1998. Although early evidence suggested that a rBCG-Llo+ strain was more adept at eliciting CD8+ T cells, the strain proved to be incapable of escaping the endosome. Thus, a limitation of this approach is that the hemolytic function of Llo, which is required for endosome escape, is only fully active at pH 5.5 and almost inactive at pH 7.0. Since Mycobacteria maintain the pH of the endosome at a value of ca. 7.0, it is logical to surmise that Llo in rBCG-Llo strains thus far reported are dysfunctional because the environment in which they are expressed is suboptimal for hemolytic activity magnitude (Geoffroy et al., Infect Immun 55(7): 1641-1646; 1987). Thus, the immune-enhancing benefit of this approach will not be realized until a strategy is developed that enables Llo to function in BCG-manipulated endosomes.
The prior art has thus far failed to provide a rBCG with an increased capacity for endosomal escape, and which can thereby increase induction of, for example, Major Histocompatibility Complex class I-restricted CD8+ cytotoxic T cell responses.